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First published on January 3, 2008; DOI: 10.1124/mol.107.042283

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Received for publication September 28, 2007.
Revised December 17, 2007.
Accepted for publication December 27, 2007.

Pharmacological and kinetic characterization of ADM1 and CGRP1 receptor reporter cell lines

Frank Wunder 1*, Annegret Rebmann 1, Andreas Geerts 1, Bernd Kalthof 1

1 Bayer Healthcare AG

* Address correspondence to: E-mail: frank.wunder{at}bayerhealthcare.com

Abstract

Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) receptors and their respective ligands play important roles in cardiovascular (patho-)physiology. Functional expression of ADM and CGRP receptors requires the presence of the calcitonin receptor-like receptor (CRLR) together with receptor-activity-modifying proteins (RAMPs). We have characterized the expression patterns of CRLR and RAMP1-3 in human cardiovascular-related tissues by quantitative PCR. We could identify high expression levels of CRLR, RAMP1 and RAMP2 in human heart and various blood vessels. RAMP3 expression in these tissues was detectable, however, at significantly lower levels. In addition, we describe here a novel, aequorin luminescence-based GPCR reporter assay that enables the real-time detection of receptor activation in living cells. In the assay system, intracellular cAMP levels are monitored with high sensitivity by utilizing a modified, heteromultimeric cyclic nucleotide-gated (CNG) channel mediating calcium influx. Gq-coupled receptor activation is detected via aequorin luminescence stimulated by calcium release from intracellular stores. Using this novel reporter assay, we established and characterized stable ADM1 and CGRP1 receptor cell lines. The peptide ligands ADM, CGRP1 and CGRP2 were characterized as potent agonists at their respective receptors. In contrast, intermedin acted as a weak agonist on both receptors and showed only partial agonism on the ADM1 receptor. Agonist activities were effectively antagonized by the receptor antagonists ADM(22-52) and CGRP(8-37). Various vasoactive ADM fragments were also characterized but showed no activity on the ADM1 receptor cell line. In addition, luminescence signal kinetics after activation of Gs- and Gq-coupled receptors were found to be markedly different.


Key words: Purinergic, Gs family, Gq/11 family, cAMP, Calcium (G Protein Coupled Signals), Ca imaging, Peptide hormones


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